Novel mutations in the FOXC1 gene in Japanese patients with Axenfeld-Rieger syndrome

Purpose Mutations in the forkhead transcription factor (FOXC1) gene have been shown to cause juvenile glaucoma associated with a variety of anterior-segment anomalies. The purpose of this study was to determine the ocular and genetic characteristics of two Japanese families with Axenfeld-Rieger syndrome (ARS). Methods Genomic DNA was extracted from the leukocytes of six members of two families with ARS. The DNA from one exon of the FOXC1 gene were amplified by polymerase chain reaction (PCR) and directly sequenced. The patients received standard systemic and ophthalmological examinations. Results Sequence analysis of the FOXC1 gene revealed a novel Ala85Pro missense mutation in Helix1 in family 1 and a deletion of 17 nucleotides (437-453) in Wing1 and Beta2 within the forkhead domain of the FOXC1 gene in family 2. This deletion predicted a loss of the forkhead domain by a premature termination of translation. These mutations segregated with the ARS phenotype in an autosomal dominant pattern. The affected individuals in family 1 had posterior embryotoxon, iris hypoplasia, corectopia with early-onset severe glaucoma, atrial septal defect, aortic stenosis, and pulmonary stenosis. The affected members in family 2 had posterior embryotoxon and iris hypoplasia with early-onset glaucoma, and systemically they had hearing loss, hypertelorism, and telecanthus. Conclusions A novel mutation in Helix1 and a novel deletion in Wing1 and Beta2 of the forkhead domain of the FOXC1 gene have been identified in two families with ARS. FOXC1 mutations cause a variety of developmental abnormalities in the anterior segment of the eye, and they also induce an elevation in intraocular pressures and early-onset glaucoma.

Dysgenesis of the anterior segment of the eye is a genetically heterogeneous developmental anomaly [1] resulting in a high risk of glaucoma. One such anomaly is Axenfeld-Rieger anomaly (ARA), which is occasionally associated with extraocular alterations such as maxillary hypoplasia, hearing loss, dental abnormalities, congenital heart defects, redundant periumbilical skin, and skeletal limb anomalies. ARA patients with these alterations are said to have "Axenfeld-Rieger syndrome" (ARS).
Nishimura et al. [2] identified a patient with primary congenital glaucoma who had chromosomal anomalies with a balanced translocation between 6p25 and 13q22. Cloning of the 6p25 breakpoint led to the identification of mutations in the FOXC1 gene (formerly called FKHL7), and they demonstrated that mutations in the FOXC1 gene were also detected in patients with Rieger's anomaly, Axenfeld's anomaly, and iris hypoplasia. These findings demonstrated that mutations in the FOXC1 gene cause a wide variety of allelic disorders of the anterior segment such as iridogoniodysgenesis anomaly (IGDA) associated with glaucoma [3].
In a family with nine affected individuals in three generations, Mirzayans et al. [4] found that ARS was associated with a Gln23Stop amino acid substitution in the FOXC1 gene. The affected individuals presented with different degrees of iris hypoplasia, displaced pupils (corectopia), and a prominent, anteriorly-displaced Schwalbe line (posterior embryotoxon). Peripheral iris strands were seen to bridge the iridocorneal angle.

METHODS
Genomic DNA was extracted from leukocytes of peripheral blood and purified by the Qiagen QIAamp Blood Kit (Qiagen, Valencia, CA). The purpose of the study and the procedures to be used were explained to all patients and an informed consent was obtained. The procedures used conformed to the tenets of the Declaration of Helsinki. This study was approved by the Tohoku University Institutional Review Board.
The proband in family 1 was a three-year-old girl who presented at three months of age with early onset severe glaucoma and who has systemic complications including atrial septal defect, aortic stenosis, and pulmonary stenosis. Her father also manifested early-onset glaucoma. The proband of the second family was a 38-year-old woman who was diagnosed with congenital glaucoma in both eyes at birth. Family 2 had five affected members and at least three affected members had similar features. Standard ophthalmic examinations were performed on all examined patients. Control subjects (52 men and 48 women; mean age 68.0±7.7 years) had IOP>21 mm Hg, normal optic discs, and no family history of glaucoma.
The forkhead domain (FHD), spanning amino acids 69-178, was amplified by polymerase chain reaction (PCR) using 0.5 µM concentration of a pair of primers in an amplification mixture (25 µl) containing 0.2 mM dNTPs and 0.5 U of ExTaq polymerase (Takara, Japan) with 30 ng template DNA. Oligonucleotide primers used for amplification of FHD of the FOXC1 gene were forward (sense) primer 5'-AAC TCC CTG GGA GTG GTG CCC TA-3' and reverse (antisense) primer 5'-CGG CTC CTT GAG GTG CAG CCT-3'. The PCR products were purified using a PCR Purification Kit (PCR Kleen Spin Columns, Bio-Rad, Hercules, CA) after loading onto a 1.2% agarose gel. Purified fragments were directly sequenced by the BigDye™ Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Foster City, CA) on an automated DNA sequencer (ABI PRISM™ 3100 Genetic Analyzer, Perkin-Elmer).

Family 1: Missense Mutation Ala85Pro:
The proband in family 1 was a three-year-old girl who presented at three months of age with hazy megalocornea, posterior embryotoxon, iris hypoplasia, corectopia with early onset severe glaucoma, atrial septal defect, aortic stenosis, and pulmonary stenosis. The horizontal by vertical corneal diameters were 13.0 by 12.5 mm in the right eye ( Figure 1) and 12.5 by 11.0 mm in the left eye. The patient underwent trabeculotomy three times in the right eye and two times in the left eye. Her father, who had posterior embryotoxon and iris hypoplasia with early-onset glaucoma, has been followed by a local ophthalmologist.
We screened the FOXC1 sequence for mutations in this proband. After direct sequencing, a heterozygous novel point mutation (G>C) was found at the first nucleotide in codon 85 which changed alanine to proline (Ala85Pro; Figure 2). This novel missense mutation was identified in two members of this family, which is consistent with an autosomal dominant inheritance pattern. The Ala85Pro mutation was not found in 100 ethnically-matched control subjects. was diagnosed with congenital glaucoma in both eyes at birth. She underwent trabeculectomy once in the right eye and trabeculotomy/goniotomy three times in the left eye. The horizontal by vertical corneal diameters were 12.5 by 12.0 mm in the right eye and 14.5 by 12.0 mm in the left eye. She had posterior embryotoxon and iris hypoplasia with early-onset glaucoma (Figure 3). Family 2 had five affected members (Figure 4) consisting of patients 1, 2, and 3 as the proband, the father of the proband, and an aunt. All affected members (patients 1, 2, and 3) had similar features such as hearing loss, hypertelorism, and telecanthus. A heterozygous novel deletion, 437-453del17, was identified in the coding region of the FOXC1 gene in two patients in this family ( Figure 5). The mutation segregated with the disease phenotype and the distribution of the affected members was consistent with an autosomal dominant inheritance pattern. The 437-453del17 mutation was not found in 100 control subjects.

DISCUSSION
The FOXC1 gene is a member of the forkhead/winged-helix family of transcription factors. These transcription factors contain a monomeric, 110 amino acid DNA binding domain, and forkhead domain (FHD). This motif was originally identified as a region of homology with the Drosophila melanogaster forkhead protein [14] and rat hepatocyte nuclear factor 3 protein (also known as Foxa3) [15]. The FHD is evolutionarily conserved and exists in a wide range of species from yeasts to humans [16]. This DNA-binding motif is a variant of the helix-turn-helix motif and consists of three helices and two large loops that form wing structures, Wing1 and Wing2 [2,6,17].
The mutations in our patients were found in the α-helix1 in the FHD, which is located at the amino acid position 85 in family 1 (Figure 6). Previous studies had noted that the NH 2and COOH-terminal boundaries of the FHD were critical for proper nuclear localization of FOXA2 and FOXF2 [18,19]. Two lesions are named the nuclear localization signal 1 (NLS1) and nuclear localization signal 2 (NLS2). The first region, NLS1, spans residues 77-93 where Ala85Pro is located. Saleem et al. [8] showed that Leu86Pro disrupts FOXC1 function severely revealing the importance of helix 1 in FOXC1 structure and function. Thus, there is a possibility that Ala85Pro reduces the DNA binding and significantly reduces transactivation.  In family 2, a deletion of 17 nucleotides (437-453) spans residues 146-151 and is located in Wing1 and Beta2 within the FHD of the FOXC1 gene. This deletion generates a truncated 298 amino acids FOXC1 protein instead of a 553 full length amino acid ( Figure 6). Therefore, this deletion would disrupt the nuclear localization signal 2 (NLS2), which contains a basic stretch of amino acids at position 169 RRRRRFKK 176 at the COOH-terminal end of the FHD. Both NLSs are necessary for FOXC1 nuclear localization. The first region, NLS1, may serve as a nuclear localization signal (NLS) accessory domain and NLS2 could be the true nuclear localization signal [20]. Wing2 was suggested to play an important role in DNA binding and the transactivation capability of FOXC1 [9]. So, a deletion of 17 nucleotides (437-453) could possibly affect the DNA-protein interaction.
A haploinsufficiency of forkhead transcription factors has been shown to cause aberrant ocular development [2,21]. Considering these facts, we propose that these mutations may affect the migration and/or differentiation of the mesenchymal cells that contribute to the anterior segment of the eye [21] and the developmental processes including embryogenesis and tissue specific cell differentiation [16,22]. Only a small numbers of studies have reported mutations in FOXC1 in the Asian population [11][12][13] thus, it is difficult to discuss extensive genotype-phenotype correlations. Kawase et al. identified four mutations: 26-47ins22, Ile91Ser, 286ins1, and Arg127His. The younger generations had iris hypoplasia with early-onset and severe glaucoma. The patient with frame-shift mutation, 26-47ins22, exhibited a more severe phenotype than patients with the other mutations [11]. Almost all of these mutations are located in the 110-amino-acid DNA binding domain and FHD, and are evolutionarily conserved and exist in a wide range of species from yeasts to humans [16]. All of the FOXC1 mutations could have the net of reducing FOXC1 transactivation [22]. In our case, frame-shift mutation, 437-453del 17, also showed high penetrance and slightly more aggressive glaucoma phenotype. Both frame-shift mutations, 26-47ins22 and 437-453del 17, make truncated proteins and haploinsufficiency may be correlated with the anterior-chamber defects of the eye.
Current medical therapies are not successful in decreasing the lower intraocular pressure or in preventing progression of glaucoma in patients with ARS. Only 18% of patients with glaucoma and either FOXC1 or PITX2 genetic defects responded to medical or surgical treatment [23]. The genotype-phenotype correlations of the FOXC1 gene may help in establishing the prognosis of the disease processes and in understanding the mechanism associated with the various anterior segment dysgenesis caused by the FOXC1 gene.  1008